This proposal deals with the unprecedented phenomenon of mRNA capping in the cytoplasm. The 5' ends of all mRNAs have an m7G `cap', and proteins that bind to the cap direct the processing, translation and fate of every transcript. The prevailing view was that caps could only be added to newly synthesized pre-mRNAs in the nucleus, and loss of the cap leads irreversibly to mRNA decay. In contrast, we identified transcripts that are stable in an uncapped state, identified a cytoplasmic complex of proteins that can restore the cap onto these transcripts, and identified a cyclical process of decapping and recapping termed `cap homeostasis' that maintains a subset of the transcriptome in an actively translating state. The process of cytoplasmic capping involves conversion of the 5'-monophosphate end of uncapped RNA to a 5'-diphosphate and the transfer of GMP from capping enzyme onto this recapping substrate. All of the enzymes needed to catalyze cytoplasmic capping are present in a single complex that assembles on Nck1, a cytoplasmic SH2/SH3 adapter protein that is best known as a transducer of tyrosine kinase signaling. The RNA 5'-kinase and capping enzyme are juxtaposed by binding to adjacent SH3 domains, and the presence of cap methyltransferase in the complex completes the list of proteins that are necessary and sufficient to affect cytoplasmic capping. Cytoplasmic capping targets are not random; they encode proteins involved in nucleotide binding, protein localization, RNA localization, and the mitotic cell cycle. The working hypothesis of this proposal is that cytoplasmic capping is a selective post-transcriptional process that functions as an amplifier of transcriptome and proteome complexity. In Aim 1 in vitro, in vivo and biochemical biological approaches will be used to characterize the 5'-kinase and its function in cytoplasmic capping. Nck1 has 4 functional domains, and classical and `top-down' proteomics and biochemical approaches will be used to identify and characterize proteins that are bound uniquely to the 1st SH3 domain and SH2 domain in the context of the cytoplasmic capping complex. Aim 2 will map the 5' ends of recapped transcripts and determine their relationship to internal cap sites identified by Capped Analysis of Gene Expression (CAGE). The resulting datasets will be mined to identify sequence and/or structural motifs that determine the location of recapped 5' ends and their role in determining target specificity. In Aim 3 ribosome profiling will be combined with positional proteomics to determine the relationship of cytoplasmic capping to translation and proteome complexity. The results will be confirmed by top-down proteomics of selected products from internally capped transcripts, and changes in subcellular distribution will be used as an assay for functional effects of downstream capping on protein diversity. In summary this work will determine the organization of the cytoplasmic capping complex, the location of recapped ends within target transcripts, and the impact of cytoplasmic capping on transcriptome and proteome complexity.